CN103435625A

CN103435625A - Red light emission rhodamine-based ionic fluorescent probe and application thereof

Info

Publication number: CN103435625A
Application number: CN2013103408003A
Authority: CN
Inventors: 王晓飞; 毛飞
Original assignee: Bao Tian Biotechnology (shanghai) Co Ltd
Current assignee: Bao Tian Biotechnology (shanghai) Co Ltd
Priority date: 2013-08-07
Filing date: 2013-08-07
Publication date: 2013-12-11
Anticipated expiration: 2033-08-07
Also published as: CN103435625B

Abstract

The invention discloses a red light emission rhodamine-based ionic fluorescent probe which is a compound with a general formula (I) as shown in the specification or a compound with a general formula (II) as shown in the specification. The invention further discloses an application of the fluorescent probe. The fluorescent probe can be used for measuring concentrations of ions in a system, a cell and a tissue, and particularly for measuring concentrations of calcium ions or magnesium ions in the system, the cell and the tissue.

Description

Red emission rhodamine ion fluorescence probe and application thereof

Technical field

The present invention relates to a class new compound, be specifically related to the fluorescence dye field, particularly class red emission rhodamine ion fluorescence probe and an application thereof, particularly its application in living things system.

Background technology

Calcium ion plays an important role in the vital movement of cell, be the indispensable important ion of the every physiological activity of body, many physiological and biochemical procedures all must have Ca as signal transmission, Muscle contraction, substance metabolism, cell eubolism, cytodifferentiation and propagation etc. ²⁺participate in.Along with people, for calcium metabolism, calcium channel, calcium acceptor and regulation and control thereof, the understanding of the transhipment of calcium and utilization and some Occurrence and development of disease relation is more and more deep, Ca in cell ²⁺concentration has become the common issue of cytology, cytobiology, Neuroscience Research in the variation in time and/or space.Therefore, need to measure more accurately on cell or ubcellular space and obtain the more suitable time of response calcium ion concn, and these be all in the situation that do not affect the biological procedures of system and carry out.So far, so-called fluorescence calcium ion probe can well meet this demand, a series of fluorescence dyes through particular design are arranged, when passing through the chelating calcium ion, these fluorescence dyes produce the variation of fluorescence spectrum character, these variations can be the changes of dye fluorescence intensity or wavelength, when also can be the two, change.Typically, change and the calcium ion concn of spectrum are linear, therefore can carry out quantitatively the concentration of calcium ion.

Have benefited from Tsien and co-worker's thereof initiative work, produced a series of fluorescent probes with different photoluminescent properties and calcium ion dissociation constant at present.Have up till now three generations's calcium fluorescent probe: the first-generation is synthetic quin-2(Biochemistry, 19:2396-2404 of early 1980s); After synthesized again the s-generation probe that can measure for the dual wavelength ratio, i.e. Indo-1 and Fura-2 (J.Biol.Chem.260:3440-3450; U.S.Pat.No.4,603,209), calcium ion probe all needs ultraviolet excitation this in two generation; Synthesized again afterwards the third generation fluorescent probe by excited by visible light, i.e. Fluo-3 (J.Biol.Chem.264:8179-8184); Synthesized again afterwards Rhod-2 (U.S.Pat.No.5,049,673), and the calcium of molecular probe company green-1 and green-2 (U.S.Pat.No.5,453,517) of calcium, the emission wavelength of these several fluorescent probes is positioned at blue light mostly, and green glow is to the gold-tinted zone.

Current business-like fluorescent probe still has shortcoming: topmost problem is that exciting with emission wavelength of they is relatively short.For example, when using short-wavelength light (UV-light) while being excited, many groups of cells branch emission blue-fluorescence, this will be attributed to so-called autofluorescence or background fluorescence.Ultraviolet excitation also likely causes cell injury, for example, to the destruction of DNA, therefore for viable cell, surveys, and the dyestuff of ultraviolet excitation is also improper.And excited by visible light Ca ²⁺the Ca of fluorescent probe and burst of ultraviolel ²⁺fluorescent probe is compared, and has lot of advantages: can effectively be excited by the equipment based on laser by great majority, comprise laser scanning co-focusing microscope and flow cytometer; Can effectively avoid the damage to cell that causes due to ultraviolet excitation and the interference of cell autofluorescence; Cell photo-damage is little, a little less than scattering of light; When the dyestuff absorption is very strong, can reduce the concentration of dyestuff, therefore can reduce the toxicity to viable cell; There is good consistency with the probe of photoactivation probe (lock cage) and other UV photoabsorption, can increase the selectivity of Multi-parameter measurement; Along with calcium ion concn increases, fluorescence intensity significantly increases, detection Ca that can be sharper ²⁺the instantaneous variation of concentration, under many circumstances, along with calcium ion concn increases, fluorescence intensity change is larger, more is conducive to the detection of calcium ion transition.Based on above some, the calcium ion fluorescent Fluo-3 of visible ray class and Rhod-2 are the significant improvement to Indo-1 and Fura-2.Thereafter Calcium Orange and the Calcium Crimson of exploitation expand to the spectrum of Fluo-3 and Rhod-2 orange-colored light especially to region of red light (U.S.Pat.No.5,453,517).U.S.Pat.No.5,453, the 517 Calcium Orange that describe, Calcium Crimson and other similar calcium ion fluorescents and former calcium ion fluorescent (Fura-2 for example, Indo-1, Fluo-3 and Rhod-2) there is basic difference, in former calcium ion fluorescent design, the calcium ion chelator part directly partly is connected with chromophoric group.The result that ion chelating agent part directly is connected with chromophoric group is that the chelating to ion produces extraordinary spectral response.Yet the design of Calcium Orange and Calcium Crimson is that the calcium ion chelator part is connected by covalently bound group with chromophoric group.Metal ion chelation agent makes spectrum lower to the chelating response of calcium ion with chromophoric non-directly being connected.

In a word, although the course that the exploitation of calcium ion fluorescent has been passed by 30 years, but still need to develop and there is high sensitivity and there is the novel agents of calcium ion fluorescent probe that long wave absorbs and long wave is launched simultaneously, need to develop a series of fluorescent probes with different calcium ionic dissociation constant simultaneously, thereby meet the demand that in system, cell and tissue, calcium ion is measured.

Summary of the invention

One of the technical problem to be solved in the present invention is to provide a kind of novel cpd (i.e. logical formula I compound), and this compound can be used as non-permeable membrane fluorescent probe;

Two of the technical problem to be solved in the present invention is to provide another kind of novel cpd (i.e. logical formula II compound), and this compound can be used as the permeable membrane fluorescent probe;

Three of the technical problem to be solved in the present invention is to provide the application of logical formula I compound, and it surveys the method for calcium in sample ion or magnesium ion as fluorescent probe.

Four of the technical problem to be solved in the present invention is to provide the application of logical formula II compound, and it is as the method for calcium ion or magnesium ion in fluorescent probe detection viable cell sample.

The invention provides a class and can be used as far infrared and excite, the fluorescent probe of far infrared transmission, it is chelating moiety that this fluorescent probe be take BAPTA or APTRA, the rhodamine ring of expansion of take is chromophoric group; The invention provides the demand of ion fluorescence probe to meet system, cell and to organize interior ion measurement with different dissociation constants; The invention provides a kind of ion fluorescence probe of the permeable membrane of measuring for ion concentration in cell; The invention provides the compartmentation problem that a kind of water-soluble probe of ion fluorescence preferably exists when well improving in cell ion measurement; The invention provides a kind of method of easy fast synthetic this type of fluorescent probe to meet business-like demand.

Before describing the present invention in detail, various terms are described with helping understanding:

Term " covalently bound group " or " L " refer to single covalent linkage herein or the covalent linkage of the series of stable that is comprised of 1-30 non-hydrogen atom (can select C, N, O, S and P), and contain a chemical active radical and be connected with target molecule.Covalently bound group can become to be grouped into binding substances with other, and for example target is partly antibody, part, biomolecules, medicine and its analogue.

Term " carrier molecule " or " W " refer to form covalent linkage between the compounds of this invention and bioactive ingredients or abiotic activeconstituents herein.These compositions include but are not limited to: amino acid, polypeptide, protein, polysaccharide, nucleosides, Nucleotide, oligonucleotide, nucleic acid, haptens, fat element, medicine, hormone, lipid, liposome, dextran, synthetic polymer, polymer particle, biomass cells, virus or its array configuration.

Term " avidity " refers to two bonding forces between molecule herein, for example, between sequestrant and metal ion or the bonding force between positively charged ion and negatively charged ion.

Term " system " refers to that take water is main component and retains the solution system of the characteristic of water herein, and herein, water solution system can refer to and add some other solvent in water, but water is still main component.

Term " cell permeable membrane " refers to and can on the compounds of this invention, connect lipophilic group through the cell walls of viable cell, thereby passes cytolemma and enter viable cell.Once enter in cell, thereby lipophilic group is hydrolyzed formation, charged group is retained in viable cell the compounds of this invention.Useful especially lipophilic group comprises acetyl-o-methyl (AM) ester and ethyl acetate, once enter in cell, they will be become charged molecule by the nonspecific esterase hydrolysis.

The direct or indirect signal that can be detected by naked eyes or instrument that term " probe response " refers to detect in sample that existence due to metal ion causes herein.Typically, probe response is optical signalling, can be the changes in distribution of wavelength, absorbs or fluorescence intensity, and scattering of light changes, fluorescence quantum yield, and fluorescence lifetime, fluorescence polarization, excite or the displacement of emission wavelength or the mixing variation of above parameter.Yet the variation of fluorescence intensity enhancing and/or fluorescence excitation or emission is the most useful.The fluorescence change that causes fluorescent probe based on ionic bond is generally due to the ground state of fluorophore or excited state variation, electron density in the ionic bond site changes, with produce fluorescent quenching after the metal target ionic bond, or have the combination of these or other factor to cause.

Term " fluorophore " refer to herein the compound primary fluorescence or with bioactive ingredients or metal ion or after enzyme metabolism with product (for example procrypsis group) in conjunction with and the change in fluorescence that produces.Thereby can be modified and be changed its solvability, spectral quality or physical properties fluorophore.Most of known fluorophore includes but are not limited to tonka bean camphor, acridine, and furans, dansyl, cyanines, pyrene, naphthalene, cumarone, quinoline, quinazolinone, indoles, benzoxazoles, and xanthene (comprising fluorescein, rhodamine etc.) etc.Strengthened solvability thereby fluorophore can modify, the viable cell permeable membrane, change Absorption and emission spectra.

Term " the metal-chelating ion " or " metal target ion " refer to herein can with existing BAPTA or APTRA and analogue thereof, any metal ion of derivative combination.Typically, these metal ions all have relative physiology or nutrition significance, for example: Na ⁺, K ⁺, Zn ²⁺, Mg ²⁺, Fe ²⁺and Ca ²+.The described metal ion of entry can be also Ga ³⁺, Tb ³⁺, La ³⁺, Pb ²⁺, Hg ²⁺, Cd ²⁺, Cu ²⁺, Ni ²⁺, Co ²⁺, Mn ²⁺, Ba ²⁺and Sr ²⁺.

Term " active group " refers to herein and can react with other chemical groups the group that forms covalent linkage, for example under synthetic reaction conditions, has the covalency activity, the point that general proxy can be connected with other substrate.Active group is a part just, for example carboxylic acid or succinimide ester, and in the present invention, the indication active group is can the covalently bound functional group of compound formation different from other.Active group generally comprises nucleophilic, parent's electricity and photoactivation group.

Term " sample " refers to any material that comprises metal ion herein.Typically, sample is viable cell or the biofluid consisted of interior living chief cell albumen or food or environmental sample (for example water sample).Sample can be the aqueous solution, cell culture, the set thing on solid or semi-solid surface, polyacrylamide gel for example, press back mark or micromatrix.

The invention describes a class novel cpd, this class novel cpd can be used as the fluorescent probe use that ionic concn is measured, this fluorescent probe comprises ligand moiety (metal ion chelation agent) and the fluorophore two portions of being combined with metalloform-selective, and directly ligand moiety partly is connected with fluorophore, when the ligand moiety of probe when certain metal ion species is combined, cause the probe response of the fluorescence parameter of fluorophore, according to this association, can obtain the information for the treatment of measured ion.Therefore, fluorescent probe of the present invention can be used for metal ion is detected, quantitative and qualitative analysis.With the similar probe of current existence, compare, a remarkable advantage of probe of the present invention is to have longer absorbing wavelength and fluorescent emission wavelength, typical, and probe emission of the present invention is red to scarlet fluorescence; The present invention has more long wavelength's probe can make the autofluorescence of living things system minimize; In addition, redness or scarlet fluorescence can with other more short wavelength's fluorescent probe combine use, for example with green or blue-fluorescence probe, combine to use and develop for multicolor fluorescence.

The present invention has also described the synthetic and using method of this type of fluorescent probe.

Metal ion chelation agent

The invention describes metal ion chelation agent, is can be combined with metal ion or any part of chelating.Typically, in conjunction with or the result of chelating be the variation that causes fluorescent signal.Metal ion described in the invention refers to but is not limited in Zn ²⁺, Mg ²⁺, Fe ²⁺and Ca ²⁺, Ga ³⁺, Tb ³⁺, La ³⁺, Pb ²⁺, Hg ²⁺, Cd ²⁺, Cu ²⁺, Ni ²⁺, Co ²⁺, Mn ²⁺, Ba ²⁺and Sr ²⁺.Generally speaking, this metal ion is generally the metal ion with physiologic meaning, comprises Zn ²⁺, Mg ²⁺, Fe ²⁺and Ca ²⁺.Further, this metal ion is Ca ²⁺.In calcium ion mensuration, the most trustworthy sequestrant is polycarboxylic acid's class sequestrant at present, BAPTA or APTRA and analogue thereof are polycarboxylic acid's class sequestrant, therefore can use BAPTA or APTRA and analogue thereof as the metal ion-chelant part, BAPTA or APTRA and analogue thereof also can be used for above-mentioned other ion of chelating.

Herein as metal-chelating part " BAPTA " be two (2-amino-benzene oxygen) ethane-N of 1,2-, N, N ', N '-tetraacethyl or its analogue, derivative (for example ester, acid amides, carboxylamine etc.), variant and binding substances, all metals or non-metal salt, part salify and hydrate.Its basic structure is as follows:

Herein as metal-chelating part " APTRA " be Ortho-Aminophenol-N, N, O-nitrilotriacetic or its analogue, derivative (for example ester, acid amides, carboxylamine etc.), variant and binding substances, all metals or non-metal salt, part salify and hydrate.Its basic structure is as follows:

In addition, also can there is following structure as the metal-chelating part:

Can be also its analogue, derivative (for example ester, acid amides, carboxylamine etc.), variant and binding substances, all metals or non-metal salt, part salify and hydrate.

Phenyl ring on BAPTA or APTRA can be replaced by one or more groups herein, thereby changes the avidity of compound and metal ion, solvability, and chemically reactive, spectral quality or other physical propertiess, but at least on a position, by chromophoric group, replaced.Typically, generally choose the contraposition of N as chromophoric the position of substitution, chromophoric group is directly or indirectly replaced phenyl ring; And vinyl dioxy (OCH ₂cH ₂o-) hydrogen atom on also can be substituted, and substituting group can be can contain active group in covalently bound dose of L(L) L-active group or L-W, " W " refers to form covalent linkage between the compounds of this invention and bioactive ingredients or abiotic activeconstituents herein.These compositions include but are not limited to: amino acid, polypeptide, protein, polysaccharide, nucleosides, Nucleotide, oligonucleotide, nucleic acid, haptens, fat element, medicine, hormone, lipid, liposome, dextran, synthetic polymer, polymer particle, biomass cells, virus or its array configuration.

Polycarboxylic acid's compound can the permeable membrane ester form enter cell, in cell, ester is the polycarboxylic acid by enzymic hydrolysis, mode that also can be by microinjection is polycarboxylic acid's transfered cell, and the polycarboxylic acid can't permeable membrane, therefore, once enter in cell, polycarboxylic acid's ion can't permeable membrane.After polycarboxylic acid and calcium binding, carboxy moiety can make the C-N key rotate after calcium is combined, thereby lone-pair electron and conjugated structure on nitrogen-atoms are broken away from, and will cause fluorophore intensity and/or spectrum to change.According to this kind of association, just can measure the concentration of intracellular calcium.

Fluorophore

Fluorophore of the present invention is partly the reporter molecules that can produce direct or indirect detectable signal relevant to the metal target ion, consequently can be used for surveying, and the metal target ion in monitoring and quantitative sample.Particularly, the present invention adopts 5 rings or 7 ring rhodamine compounds as fluorophore.

Therefore, from saying on the one hand, the non-permeable membrane fluorescent probe of the present invention can use general structure (I) to mean:

Wherein, R ₂, R ₃, R ₄independent mutually, R ₂, R ₃, R ₄for hydrogen, fluorine, chlorine, bromine, iodine, cyano group, hydroxyl, nitro, SO ₃ ^-, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group; Perhaps R ₂, R ₃, R ₄for by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, SO ₃ ^-, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group;

R ₅for methyl ,-CH ₂cO ₂ ^-,-L-W or

Or-CH ₂cONR ¹r ², R wherein ¹, R ²independent mutually, R ¹, R ²hydrogen, C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, or by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, SO ₃ ^-, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group, or R ¹, R ²interconnection, directly be connected or pass through oxygen, nitrogen, sulphur, silicon is connected to form saturated or unsaturated five-ring or six-ring;

R ₉-R ₁₂independent mutually, R ₉-R ₁₂for hydrogen, fluorine, chlorine, bromine, iodine, cyano group, hydroxyl, nitro, sulfonic group, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group; Perhaps R ₉-R ₁₂for by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, sulfonic group, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group; Or R ₉-R ₁₂in any one be-L-W;

R ₆for Jia Ji Huo – CH ₂sO ₃ ^-;

R ₇for hydrogen, methyl, ethyl, or and R ₈be connected to form saturated or undersaturated five-ring or six-ring or be-L-W;

R ₈for hydrogen, fluorine, chlorine, bromine, iodine, cyano group, SO ₃ ^-, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group; Perhaps, R ₈for by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, SO ₃ ^-perhaps carboxylic acid C ₁-C ₆the C that ester replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group; Perhaps R ₈for with R ₇be connected to form saturated or unsaturated five-ring or six-ring;

L is covalently bound group;

W is carrier molecule, preferably dextran;

Each M is mutually independently positively charged ion, is selected from hydrogen, potassium, sodium, ammonium, lithium or caesium, d in order to equilibrium compound the number of electronegative required M.

Above-mentioned C ₁-C ₁₈alkyl refer to the alkyl of the straight or branched with 1-18 carbon atom, for example: methyl, ethyl, propyl group, sec.-propyl, the tertiary butyl, heptyl, octyl group etc.

Above-mentioned C ₁-C ₁₈alkoxyl group refer to the alkoxyl group of the straight or branched with 1-18 carbon atom, for example: methoxyl group, oxyethyl group, propoxy-, isopropoxy, tert.-butoxy, heptan oxygen base, octyloxy etc.

Above-mentioned cyano group, molecular formula is :-CN.

Above-mentioned hydroxyl, molecular formula is :-OH.

Above-mentioned nitro, be called again nitroxyl, and molecular formula is :-NO ₂.

Above-mentioned sulfonic group, also referred to as sulfo group, molecular formula is-SO ₃h.

Above-mentioned carboxylic acid, molecular formula is :-COOH.

Above-mentioned carboxylate salt, for example :-COOK.

Above-mentioned carboxylic acid C ₁-C ₆ester, have 1-6 carbon atom carboxylicesters (molecular formula of carboxylicesters is: RCOOR '), for example: COOEt.

As preferred technical scheme, R ₂, R ₃, R ₄be all hydrogen;

R ₅for methyl ,-CH ₂cO ₂ ^-or

Or

Herein, R ₉, R ₁₁, R ₁₂independent mutually, R ₉, R ₁₁, R ₁₂for hydrogen, fluorine or chlorine, R ₁₀for hydrogen, methyl, nitro, fluorine or chlorine.

As preferred technical scheme, R ₇for methyl or ethyl, R ₈for hydrogen.

As preferred technical scheme, R ₇and R ₈be joined together to form six-ring.

The fluorescent probe of the non-permeable membrane of cell exists by this way, the concentration of metal ions that can be used in testing environment, convenient more a lot of than the concentration that is used for detecting metal ion in viable cell, the non-dialytic fluorescent probe of cell can be also that the load of cell permeable membrane fluorescent probe enters the then result after nonspecific esterase hydrolysis in cell of cell.The dialytic fluorescent probe of cell is converted into the non-dialytic fluorescent probe of cell through this kind of mode, therefore can be retained in cell and is used for measuring the concentration of metal ions in cells.

On the other hand, the fluorescent probe that can be used for the detection of viable cell intermediate ion concentration of permeable membrane of the present invention has following general structure (II):

Wherein, the R of replacement ₂, R ₃, R ₄independent mutually, R ₂, R ₃, R ₄for hydrogen, fluorine, chlorine, bromine, iodine, cyano group, hydroxyl, nitro, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group, or R ₂, R ₃, R ₄for by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group;

R ₅for methyl ,-CH ₂cO ₂r ₁or

Or-CH ₂cONR ¹r ², R wherein ¹, R ²independent mutually, be hydrogen atom, C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group; Perhaps R ¹, R ²interconnection, directly be connected or pass through oxygen, nitrogen, sulphur, silicon is connected to form saturated or unsaturated five-ring or six-ring;

R ₉-R ₁₂independent mutually, be hydrogen, fluorine, chlorine, bromine, iodine, cyano group, hydroxyl, nitro, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group, or R ₉-R ₁₂for by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group;

R ₆for methyl;

R ₇for hydrogen, methyl, ethyl, or and R ₈be connected to form saturated or undersaturated five-ring or six-ring;

R ₈for hydrogen, fluorine, chlorine, bromine, iodine, cyano group, C ₁-C ₁₈alkyl, or C ₁-C ₁₈alkoxyl group; Perhaps R ₈for by fluorine, chlorine, bromine, or the C that replaces of iodine ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group; Perhaps R ₈for with R ₇be connected to form saturated or unsaturated five-ring or six-ring;

R ₁be selected from methyl, ethyl or acetyl-o-methyl;

Ψ is negatively charged ion, and in order to equilibrium compound, institute is positively charged.

Above-mentioned cyano group, molecular formula is :-CN.

Above-mentioned hydroxyl, molecular formula is :-OH.

Above-mentioned carboxylic acid, molecular formula is :-COOH.

Above-mentioned carboxylate salt, for example :-COOK.

Carboxylic acid can be used acetyl-o-methyl (AM) to be modified herein, makes carboxy moiety not containing electric charge, thereby can make fluorescent probe permeates cell membranes of the present invention enter cell, be i.e. R ₁for-CH ₂oCOCH ₃, R ₁can be also methyl, ethyl.

For metal ion chelation agent APTRA and analogue thereof, the replacement R on phenyl ring ₂, R ₃, R ₄independent mutually, can be hydrogen atom, halogen (halogen refers to fluorine atom herein, chlorine atom, bromine atoms, iodine atom), cyano group, hydroxyl, nitro, nitroso-group, thioether, SULPHURYL CHLORIDE; C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, or by be halogen, carboxylic acid, carboxylate salt, the C of carboxylicesters replacement ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group ,-L ,-L-active group ,-L-W.Typically, R ₂, R ₃, R ₄for halogen or hydrogen atom, furthermore, R ₂, R ₃, R ₄be preferably hydrogen.

R ₅be preferably methyl ,-CH ₂cO ₂r ₁or

Or

Herein, R ₉, R ₁₁, R ₁₂independent mutually, be hydrogen, fluorine or chlorine, R ₁₀for hydrogen, methyl, nitro, fluorine or chlorine;

R ₁for acetyl-o-methyl.

For metal ion chelation agent BAPTA and analogue thereof, the replacement R on phenyl ring ₂, R ₃, R ₄done definition in APTRA, furthermore, R ₉-R ₁₂independent mutually, can be hydrogen atom, sulfonic group, alkylsulfonyl, amino, cyano group, hydroxyl, nitro, alkyl or halogen (halogen refers to fluorine atom herein, chlorine atom, bromine atoms, iodine atom), C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, or by halogen, carboxylic acid, carboxylate salt, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group ,-L ,-L-active group ,-L-W.Typically, R ₉, R ₁₁, R ₁₂preferred hydrogen atom or alkyl, further, R ₉, R ₁₁, R ₁₂preferred hydrogen atom; R ₁₀be preferably halogen, nitro, amino, hydrogen atom, alkyl ,-L-active group, or-L-W, further, alkyl preferable methyl, the preferred fluorine atom of halogen, chlorine atom.

Fluorophore of the present invention is 5 rings or 7 ring rhodamine compounds, as shown in general structure (I) and general structure (II), and R wherein ₆for alkyl, substituted alkyl Huo – CH ₂sO ₃ ^-, further, when sequestrant is partly non-permeable membrane free love acid, R ₆for alkyl, substituted alkyl Huo – CH ₂sO ₃ ^-, typical, R ₆preferred alkyl Huo – CH ₂sO ₃ ^-, alkyl is preferably methyl; And when sequestrant partly is the permeable membrane ester, R ₆for alkyl, the alkyl preferable methyl;

R ₇for hydrogen atom, alkyl or and R ₈be connected to form saturated or undersaturated five-ring or six-ring or be-L-W; Furthermore, R ₇preferred hydrogen atom, methyl, ethyl or and R ₈be connected to form saturated or undersaturated five-ring or six-ring; R ₇more preferably methyl or ethyl.

R ₈can be hydrogen atom, fluorine atom, chlorine atom, bromine atoms, iodine atom, cyano group, SO ₃ ^-, or C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, C herein ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group can be further by fluorine atom, chlorine atom, bromine atoms, iodine atom, carboxylic acid, carboxylate salt, SO ₃ ^-or carboxylic acid C ₁-C ₆ester replaces; Perhaps with R ₇be connected to form saturated or unsaturated five-ring or six-ring; Further, R ₈be preferably hydrogen atom, or and R ₇be connected to form saturated or unsaturated five-ring or six-ring;

For whole fluorescent probe molecule, the dM in general structure (I), wherein each M is mutually independently positively charged ion, is selected from hydrogen, potassium, sodium, ammonium, lithium or caesium, d in order to equilibrium compound the number of electronegative required M.In general structure (II), Ψ is negatively charged ion, the positively charged negatively charged ion for the balance dyestuff.Ψ has biocompatibility, and suitable negatively charged ion includes but not limited to: halide-ions, sulfate radical, phosphate radical, perchlorate, tetrafluoroborate and hexafluoro-phosphate radical.Typically, negatively charged ion is chlorion or iodide ion (just for example).

Synthetic

The invention provides a kind of method of synthetic the compounds of this invention, the preparation method of the compounds of this invention roughly is divided into two parts.At first need to form the metal ion chelation agent part, thereby then need metal ion chelation agent is partly modified and formed active function groups, thereby active function groups partly is connected and forms whole compound with chromophoric group.

As general structure (I) and the described metal ion chelation agent part of general structure (II), the replacement of different nature of phenyl ring nitrogen and oxygen has material impact to the sequestering power of final sequestrant, therefore, for different metal target ions, select suitable substituting group necessary.BAPTA has good calcium ion selective, and its phenyl ring nitrogen-atoms is replaced by methyl bromoacetate, and APTRA has good magnesium ion selectivity, and its nitrogen-atoms and Sauerstoffatom are all replaced by methyl bromoacetate.Select suitable replacement precursor extremely important for preparing the target compound with different dissociation constants.

A lot of documents reach synthetic all have description, U.S.Pat.No.4,603,209 to the selection of the sequestrant of different metal ion; U.S.Pat.No.5,049,673; U.S.Pat.No.4,849,362; U.S.Pat.No.5,453,517; U.S.Pat.No.5,501,980; U.S.Pat.No.5,459,276; U.S.Pat.No.5,501,980; U.S.Pat.No.5,459,276; U.S.Pat.No.5,516,911; The method of describing in these patents may be applicable to for the chelating moiety intermediate in synthetic the compounds of this invention.

Synthesizing at document (G.Grynkiewicz of metal ion chelation agent BAPTA tetramethyl ester; M.Poenie and R.Y.Tsien1985; J.Biol.Chem.260; description had been arranged 3440-3450), suitably modified on this basis again and the sequestrant BAPTA part that is used in synthetic the present invention; Synthesizing at document (Bert Metten of metal ion chelation agent APTRA trimethyl, Mario Smet, Noel Boens, Wim Dehaen, synthesis2005,11, description had been arranged in 1838-1844), can suitably modify on this basis and the sequestrant APTRA part that is used in synthetic the present invention.

The synthetic universal method of cluck ton class dyestuff (for example fluorescein and rhodamine) be by 2 equivalent Resorcinols (for fluoresceins synthetic) and meta-aminophenol (synthetic for rhodamine) with containing carbonyl moiety for example the phthalic acid derivatives condensation obtain corresponding cluck ton class dyestuff.Perhaps, in bibliographical information, the method for synthetic cluck ton class dyestuff was aldehyde compound condensation under strong acidic environment of 2 equivalent Resorcinols (for synthesizing of fluoresceins) and meta-aminophenol (synthetic for rhodamine) and 1 equivalent in the past, for example can in 70% sulphuric acid soln, reflux and obtain dihydro xanthene dye intermediate, then then oxidation obtains final dye, unfortunately, present method can't prepare final product when preparing the compounds of this invention.Traditional calcium ion probe based on xanthene dye be by xanthone and bromine replace the BAPTA tert-butyl ester use butyllithium at low temperatures condensation prepare, same, this kind of method can't be suitable for the compounds of this invention, because have the different xanthones that replace, is difficult to prepare.Therefore, the present invention develops a kind of method of new this kind of dyestuff of preparation.

Generally will be through peroxidation step after the Chelate Intermediate condensation that fluorophore precursor and formyl radical replace.The dihydro condensation product obtained can first pass through separation or be directly used in oxidising process, can select atmospheric oxidation or use typical chemical oxidizing agent (for example DDQ or tetrachlorobenzoquinone) oxidation, for some chromophoric grouies, under acidic conditions, the oxidation meeting is strengthened.These gentle oxidizing conditions are applicable to the different fluorescent probes that replace.

After condensation, the modifying method of the modification of metal ion-chelant part and known fluorescent probe is similar.By nitroreduction, be for example amino, amino can independently exist also and can directly or indirectly be connected with carrier molecule, for example dextran is connected with amino by covalently bound group, or carbonyl is converted into to cyano group, perhaps carboxylic acid is converted into to ester, comprises the acetyl-o-methyl ester.The salt that fluorescent probe of the present invention obtains or counterion also can pass through ion exchange resin, and selection is separated out and these currently known methodss that alkalize are converted into other salt.

After condensation, the modification of rhodamine ring also has a lot of currently known methodss, for example sulfonation, sulfonating chlorinating, after sulfonating chlorinating also can with L, the L-active group, L-W connects, or uses suitable halogenating agent to carry out halogenation, for example uses bromine to carry out bromination.The undersaturated ring of rhodamine also can carry out hydrogenation and obtain the saturated rings derivative.

May use oxygenant or reductive agent during the rhodamine fluorescent probe is synthetic.Suitable reductive agent comprises hydroborates, aluminum hydride compound, hydrogen/catalyzer, hyposulfite.The selection of reductive agent or oxygenant must be considered the relevant substituting group of chelating moiety.The oxygenant that is applicable to the dihydro rhodamine comprises oxygen/catalyzer, nitrogen protoxide, peroxynitrite, dichromate, triphenyl carbon cation, DDQ and tetrachlorobenzoquinone.The dihydro rhodamine also can pass through electrochemistry or oxydasis, comprises peroxidase and superoxide or nitric oxide production co-oxidation.

Application

The fluorescent probe that the present invention describes has comprised the detection of sample intermediate ion concentration and/or quantitative.Can be for conducting with the signal of calcium ion pulse correlation in the monitoring bio system.Generally speaking, probe sequestrant of the present invention partly contains free acid or with salt form, can be used for extracellular space, cytoplasm, the vascular tissue of plant and animal, biological fluid is blood and urine for example, fermentation media, environmental sample is water for example, soil, the mensuration of waste water and seawater and chemical reaction intermediate ion level.On the other hand, probe sequestrant part of the present invention exists with ester-formin, and for example the AM ester, can be used for monitoring intracellular calcium or magnesium ion.Optical probe is for the qualitative of metal ion or quantitatively have vital role, especially for viable cell.Fluorescent probe can be continuously or the optical signalling of discontinuity detection viable cell or the solution that contains metal ion.

In another aspect of this invention, provide a kind of method of above-mentioned logical formula I compound as fluorescent probe detection calcium in sample ion or magnesium ion of applying, comprise following steps:

1) sample contacts with logical formula I compound claimed in claim 1;

2) use the light source activation sample of suitable wavelength, and;

3) survey the fluorescent emission of sample.

As preferred technical scheme, described logical formula I compound, in logical formula I, R ₂, R ₃, R ₄be all hydrogen; R ₅for methyl ,-CH ₂cO ₂ ^-or

Or

Herein, R ₉, R ₁₁, R ₁₂independent mutually, be hydrogen, fluorine or chlorine, R ₁₀for hydrogen, methyl, nitro, fluorine or chlorine.

As preferred technical scheme, in step 1), if calcium ion or magnesium ion are positioned at the cell of sample, described logical formula I compound is by the microinjection transfered cell.

As preferred technical scheme, in step 1), if calcium ion or magnesium ion are positioned at the extracellular of sample, logical formula I compound directly contacts with sample.

As preferred technical scheme, in step 3), described detection is at fluorescent microscope, on microplate reader or flow cytometer device, carries out.

In another aspect of this invention, provide a kind of method of above-mentioned logical formula II compound as calcium ion or magnesium ion in fluorescent probe detection viable cell sample of applying, comprise following steps:

1) the described logical formula II compound of cell sample and claim 5 is contacted;

2) the culturing cell sample is at least 5 minutes;

3) use the light source activation sample of suitable wavelength, and;

4) survey the fluorescent emission of sample.

As preferred technical scheme, described logical formula II compound, in logical formula II, R ₂, R ₃, R ₄complete is hydrogen;

R ₅for methyl ,-CH ₂cO ₂r ₁or

Or

R ₁for acetyl-o-methyl.

As preferred technical scheme, described cell sample is cell culture, or the cell tissue culture.

As preferred technical scheme, in step 4), described detection is at fluorescent microscope, on microplate reader or flow cytometer device, carries out.

Use fluorescent probe of the present invention, carrying out the variation of fluorescence intensity according to the variation of metal ions in cells is comparatively sensitive scheme.Therefore, should select suitable excitation wavelength to observe ionic concn change and cause the changing conditions of fluorescence reading.Generally speaking, probe need to excited near its absorption peak wavelength and its fluorescence need to be controlled near the maximum emission peak wavelength.Many biological organizations, for example plant tissue and cerebral tissue, tend to have intrinsic or background fluorescence or be called autofluorescence.Typical autofluorescence arrives green wavelength region or the wavelength region from 400nm to 500nm in blueness.Because probe of the present invention has red and scarlet fluorescence area, can be therefore the minimum interference of autofluorescence.

Particularly, the compounds of this invention as general structure (I) thus described fluorescent probe can directly contact with target sample or enter cell by microinjection forms the mark mixture; Thereby then the mark mixture being cultivated to the sufficiently long time makes object ion and the abundant combination of the compounds of this invention, thereby form culturing mixt; Use suitable light source to be excited culturing mixt, obtain exciting mixture; Record excites the fluorescent emission of mixture, by recording the fluorescent emission comparison of compound spectral quality therewith, and with ionic concn between associated, just can calculate the relevant information of ionic concn.

The compounds of this invention, as the described calcium ion fluorescent of general structure (II), can be used known method to see Tsien et al., J.CELL Biol.94,925 (1982) and Tsien et al., Nature, 295,68 (1982).By and large, cell is cultivated with the acetyl oxygen methyl esters of fluorescent probe of the present invention, then washing.Typically, the load efficiency of fluorescent probe is generally at 10%-30%, and the load level of final fluorescent probe is approximately 10-50 μ M.Cell with fluorescent probe load of the present invention after, the concentration of intracellular calcium just can be determined.

Survey the selection of calcium ion method, along with the character of fluorescent probe character and system, tissue and cell changes, preferred Detection Techniques are such as fluorescent microscope, on microplate reader and flow cytometer device, carry out.

The accompanying drawing explanation

Fig. 1 is the fluorescence spectrum figure of compound 3 under various calcium ion concns that the embodiment of the present invention 3 makes;

The calcium ion titration experiments schematic diagram data that Fig. 2 is the compound 3 that makes of the embodiment of the present invention 3;

Fig. 3 is the fluorescence spectrum figure of compound 2 under various calcium ion concns that the embodiment of the present invention 2 makes;

The calcium ion titration experiments schematic diagram data that Fig. 4 is the compound 2 that makes of the embodiment of the present invention 2;

Fig. 5 is the fluorescence spectrum figure of compound 6 under various calcium ion concns that the embodiment of the present invention 6 makes;

The calcium ion titration experiments schematic diagram data that Fig. 6 is the compound 6 that makes of the embodiment of the present invention 6;

Fig. 7 is the fluorescence spectrum figure of compound 5 under various calcium ion concns that the embodiment of the present invention 5 makes;

The calcium ion titration experiments schematic diagram data that Fig. 8 is the compound 5 that makes of the embodiment of the present invention 5;

The experimental result schematic diagram that Fig. 9 is the embodiment of the present invention 13, wherein A-C is Rhod-2, compound 2b dyestuff and compound 12 dyestuffs are at the intracellular development schematic diagram of HeLa.D-F is Rhod-2 after using digitonin to process, and compound 2b dyestuff and compound 12 dyestuffs are at the intracellular development schematic diagram of HeLa.G-I is after using Triton X-100 to process, Rhod-2, and compound 2b dyestuff and compound 12 dyestuffs are the intracellular development schematic diagram of HeLa (insert pictures is presented at the phase contrast fluorography of same developing regional inner cell).

Embodiment

The following examples can make the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.

Further describe the synthetic method of rhodamine fluorescent probe of the present invention below in conjunction with example.

Embodiment 1: compound 1 synthetic:

5.75g(0.01mol) 5-methyl-5 '-formyl radical-BAPTA tetramethyl ester (Grzegorz Grynikiewicz, Martin Poenie, Roger Y.Tsien.The journal of biological chemistry, 1985, 260 (6) 3440-3450) and 4.35g(0.02mol) 7-hydroxyl-1-ethyl-2, 2, 4-trimethylammonium-1, 2-dihydroquinoline (US5750409) is dissolved in 25ml1, in the 4-dioxane, add 2.73g(0.02mol) Zinc Chloride Anhydrous, under nitrogen protection, room temperature reaction is 5 hours, use ethyl acetate and water to be extracted, the dilute hydrochloric acid washing, the saturated common salt water washing, anhydrous sodium sulfate drying, after revolving steaming, product is dissolved in 20ml methyl alcohol, adding 4.91g(0.02mol) tetrachlorobenzoquinone (P-Chloranil) carries out oxidation, after spending the night, directly with neutral alumina, adsorb, take neutral alumina as stationary phase, take methylene dichloride: methyl alcohol=95:5 as moving phase, carry out column chromatography, obtain product 2.3g, 2.3g column chromatography product is dissolved in 20ml methyl alcohol, the sodium iodide that adds 1.5eq, after stirring 1-2h, there is solid to separate out, filter to obtain 1.95g compound 1.

Compound 1: ¹h NMR:1.1-1.45 (m, 6H), 1.63/1.65/1.69/1.17 (s, 12H), (1.91/1.93 s, 6H), 2.26 (s, 3H), (3.4-4.1 m, 4H), 3.62 (s, 12H), 4.31 (s, 8H), (4.5 m, 4H), 5.75/6.12/6.32/7.51/6.14/6.16/6.66/7.31 (s, 8H), 6.67-7.35 (m, 4H).

Embodiment 2: compound 2, and 2a, 2b's is synthetic:

Compound 1(100mg, by embodiment 1, made) be suspended in 1:1 methanol/water (V/V) (10m L), ice bath is cooling slowly adds 200mg KOH down in suspension, at room temperature stir 12-24 hour, 50m L water dilution for the aqueous solution, neutralize pH=3-5 with the 2-3mL concentrated hydrochloric acid, the solid of separating out is filtered, obtain free acid compound 2.

Compound 2: ¹h NMR:1.1-1.45 (m, 6H), 1.17/1.46/1.97/2.21 (s, 18H), 2.26 (s, 3H), (3.4-4.1 m, 4H), 4.31 (s, 8H), 4.5 (t, 4H), (5.45/5.56/6.12/6.32/6.75/6.83/7.51 s, 8H), 6.67-7.35 (m, 4H), 11 (s, 4H).

The 100mg compound 2 use KOH aqueous solution are converted into to sylvite, then wash with water on Sephadex LH-20 by chromatogram, obtain 30mg compound 2a after freeze-drying.

Compound 2a: ¹h NMR:1.1-1.45 (m, 6H), 1.17/1.46/1.97/2.21 (s, 18H), 2.26 (s, 3H), (3.4-4.1 m, 4H), 4.31 (s, 8H), 4.5 (t, 4H), 5.45/5.56/6.12/6.32/6.75/6.83/7.51 (s, 8H), 6.67-7.35 (m, 4H).

Compound 2(60mg) at room temperature be dissolved in 3m L dry DMF 70 μ L BrCH ₂oCOMe is dissolved in 2m L DMF, and it is slowly added in the solution of compound 10 under stirring in water bath.Then 130 μ L DIPEAs (iPrNEt) are slowly added in said mixture.After adding, mixture at room temperature stirs 24-36 hour.Reaction mixture is poured in frozen water, and Precipitation is arranged, and precipitation is filtered and washed with water, will after solid drying, with silica gel, through column chromatography, obtain sterling, obtains compound 2b.

Compound 2b: ¹h NMR:1.1-1.45 (m, 6H), 1.17/1.46/1.97/2.21 (s, 18H), (2.2 s, 12H), 2.26 (s, 3H), (3.4-4.1 m, 4H), 4.31 (s, 8H), 4.5 (t, 4H), (5.45/5.56/6.12/6.32/6.75/6.83/7.51 s, 8H), 6.87 (s, 8H), 6.67-7.35 (m, 4H).

Embodiment 3: compound 3 synthetic:

With 5-methyl-5 '-formyl radical-BAPTA tetramethyl ester and 7-hydroxyl-1-methyl-2,2,4-trimethylammonium-1,2-dihydroquinoline (US6372907, US5750409) is raw material, according to the synthetic method of embodiment 1, obtains compound 3, yield: 23%.

Compound 3: ¹h NMR:0.9/1.1/1.46/2.26/3.06 (s, 21H), 1.91/1.93 (s, 6H), 4.31 (s, 8H), (4.5 t, 4H), 5.45/5.75/6.12/6.32/7.38 (s, 6H), 6.75/6.83 (s, 2H), 6.67-7.35 (m, 4H), 11 (s, 4H).

Synthesizing of embodiment 4:5 '-formyl radical-5-nitro-BAPTA tetramethyl ester (compound 4)

2.31g(4.00mmol) 5-nitro BAPTA tetramethyl acid esters (R.Pethig, M.Kuhn, R.Payne, E.Adler, T.-H.Chen, L.F.Jaffe.Cell Calcium, 1989,10,491-498) (4.00mmol), pyridine 388mg(0.4m L) be dissolved in 4.0m L (50mmol) DMF, be cooled to 0 ℃, drip POCl ₃3m L (32mmol), after dropwising, remain on 0 ℃ of 5-10 minute, and then heating 1-2 hour under 65 ℃, disappear to arriving raw material by TLC plate detection reaction.Reaction after finishing is poured into water reaction mixture.Be extracted with ethyl acetate, organic layer water and saturated common salt water washing, anhydrous magnesium sulfate drying, revolve steaming and can obtain white solid, and compound 4.

Compound 4: ¹h NMR:3.68 (s, 12H), 4.2 (s, 8H), 4.56 (t, 4H), 6.4-7.7 (d, 4H), 7.26 (s, lH), 7.9 (d, lH), 9.61 (s, 1H).

Embodiment 5: compound 5 synthetic

With compound 4(embodiment 4, make) and 7-hydroxyl-1-ethyl-2,2,4-trimethylammonium-1,2-dihydroquinoline (US5750409) is raw material, according to the synthetic method of embodiment 1, obtains compound 5, yield: 15%.

Compound 5: ¹h NMR:1.1-1.45 (m, 6H), 1.63/1.65/1.69/1.17 (s, 12H), 1.91/1.93 (s, 6H), (3.4-4.1 m, 4H), 4.31 (s, 8H), 4.5 (m, 4H), (5.43/5.75/6.12/6.32/6.75/7.51/7.62 s, 8H), 6.8-7.7 (d, 4H), 11(s, 4H).

Embodiment 6: compound 6 synthetic

With 5-fluoro-5 '-Jia Xian Ji – BAPTA tetramethyl ester (Barathbiosciences, Inc., CA, BBS1306) and 7-hydroxyl-1-ethyl-2; 2,4-trimethylammonium-1,2-dihydroquinoline (US5750409) is raw material; according to the synthetic method of embodiment 1, obtain compound 6, yield: 10%.

Compound 6: ¹h NMR:1.1-1.45 (m, 6H), 1.63/1.65/1.69/1.17 (s, 12H), 1.91/1.93 (s, 6H), (3.4-4.1 m, 4H), 4.31 (s, 8H), 4.5 (m, 4H), (5.43/5.75/6.12/6.32/6.75/6.96/7.51 s, 8H), 6.67-7.35 (d, 4H), 11(s, 4H).

Embodiment 7: compound 7 synthetic

With 4-formyl radical-2-ethoxycarbonyl methoxyl group-N, two (ethoxycarbonylmethyl group) aniline of N-(Bert Metten, Mario Smet,

wim Dehaen, Synthesis, 2005,11,1838-1844) with 1,3,3-trimethylammonium-6,7-dihydro-3H, 5H-pyrido [3,2,1-ij] quinoline-8-alcohol is raw material, according to the synthetic method of embodiment 1, obtains compound 7.

Compound 7: ¹h NMR:1/1.41 (s, 12H), 1.4-4.19 (m, 12H), (1.97 s, 3H), 2.21 (s, 3H), (4.29 s, 4H), 4.68 (s, 2H), 5.43 (s, 2H), (6.15/6.75/7.38 s, 3H), 6.76/7.24 (d, 2H), 13.03(s, 3H).

Embodiment 8: compound 8 synthetic

With 4-formyl radical-2-methoxyl group-N, N-two (ethoxycarbonylmethyl group) aniline (Bacci, James P.; Kearney, Aaron M.; Van Vranken, David L.Journal of Organic Chemistry, 2005, vol.70, #22p.9051-9053) and 1,3,3-trimethylammonium-6,7-dihydro-3H, 5H-pyrido [3,2,1-ij] quinoline-8-alcohol is raw material, according to the synthetic method of embodiment 1, obtains compound 8.

Compound 8: ¹h NMR:1.1 (s, 6H), 1.46 (s, 6H), (1.97 s, 3H), 2.21 (s, 3H), (1.4-4.19 m, 12H), 3.84 (s, 3H), (4.29 s, 4H), 5.43 (s, 2H), (6.15/6.75/7.38 s, 3H), 6.76-7.24 (d, 2H), 11 (s, 2H).

Embodiment 9: compound 9 synthetic

The 8ml vitriol oil is cooled to 0 ℃, 1mmol raw material (compound 1, by embodiment 1, made) add in the vitriol oil in batches, mixture stirs 2h and then is warmed up to gradually room temperature continuation stirring 2 days under 0 ℃, it is splashed in 10ml dioxane and 300ml ether, the solid of separating out is through filtering, filter cake is scattered in water, with solid sodium bicarbonate, neutralize, after filtering, filtrate is carried out column chromatography with silica gel after revolving steaming, moving phase is acetonitrile: water=80:20, the product obtained is converted into sylvite with the KOH aqueous solution, then cross post with LH-20, wash with water, obtain compound 9 after the product freeze-drying obtained.

Compound 9: ¹h NMR:1.15 (t, 6H), 1.3 (s, 6H), (1.46 s, 6H), 2.34 (s, 3H), (2.4 m, 4H), 4.07 (s, 4H), (4.29 s, 8H), 4.56 (t, 4H), (5.45 s, 2H), 6.17/6.33/6.99/7.38/6.75/6.83 (s, 6H), 6.33-7.4 (t, 4H).

Embodiment 10: the preparation of compound 10 and compound 11

The preparation of compound 10:

1.77mmol the precursor of compound 5 (being the tetramethyl ester-formin) is dissolved in 20ml HOAc, is heated to backflow, divides 5 batches and adds the 1g reduced iron powder to be reduced, reduction finishes, and uses diatomite to be filtered, and filtrate is used ethyl acetate and water to be extracted.The saturated common salt water washing, anhydrous sodium sulfate drying, outstanding steaming obtains product.The product 0.13mmol that above-mentioned reaction is obtained is suspended in 10ml acetone (acetone), adds 10 μ l(0.14mmo under 30 ℃) thiophosgene (CSCl ₂) stir 30min, reaction finishes after rear overhang steams to put into the sherwood oil stirring and obtains product, and compound 10.

Compound 10: ¹h NMR:1.1-1.45 (m, 6H), 1.1/1.46 (s, 12H), (1.91/1.93 s, 6H), 3.4-4.1 (m, 4H), (3.62 s, 12H), 4.31 (s, 8H), (4.5 t, 4H), 5.75/6.12/6.32/7.51 (s, 4H), (6.14/6.16 s, 2H), 6.66/6.75 (s, 2H), 6.67-7.35 (m, 4H).

The preparation of compound 11:

0.028mmol BAPTA lsothiocyanates (compound 10) is dissolved in 1ml DMF, pours this solution into 0.18g(2.6 μ mol) 70, the 2ml DMSO solution (NH of the glycosaminoglycan of 000MW ₂-dextran/DMSO) in, along with the intensification dextran can become clear solution, above-mentioned lsothiocyanates is added wherein, lsothiocyanates can react with dextran, under room temperature, stirs and spends the night.This binding substances adds in the acetone of 100ml rapid stirring, filters and obtains solid, and this solid is dissolved in the 10ml deionized water again.

By above-mentioned methyl esters 1:1(V/V) the first alcohol and water dissolved, add the KOH hydrolysis of 10eq, dextran solution is regulated to its pH to 12.0 stirring 14 hours.Then using HCl to neutralize pH is 8.0, uses 12-14, and the dialysis membrane of 000MW is dialysed to hydrolysate with deionized water, needs about 3 days.Then the solution freeze-drying is obtained to solid product, compound 11.

Compound 11: ¹h NMR:1.1-2.21 (m ,-CH ₃), 3.3-4.1 (m, dextran H C ₂-C ₆, ), 4.5 (m ,-OCH ₂-, ), 5.01 (m, dextran H C ₁), 5.45 (m,

), 6.67-7.35 (m, ArH).

Embodiment 11: the preparation of compound 12

Compound 3(30mg, 0.034mmol) at room temperature be dissolved in 2ml DMF, add 120 μ L(0.68mmol) DIPEA and 70 μ L(0.68mmol) the brooethyl acetic ester.Mixture at room temperature stirs and spends the night, and solvent is carried out to vacuum concentration.The residuum silica gel column chromatography, used ethyl acetate and sherwood oil system to carry out chromatography and obtain product 12 for purple solid 12mg, and compound 12.

Compound 12: ¹h NMR:0.9-1.4 (s, 12H), 0.9 (s, 3H), 1.97 (s, 3H), 2.21 (s, 3H), 2.19 (s, 12H), (2.28 s, 3H), 3.02(s, 3H), (4.5 m, 4H), 4.31 (s, 8H), (5.69/6.66/6.12/6.32 s, 4H), 6.66-7.4 (d, 4H), (6.87 s, 8H), 5.43/5.45/6.17/6.85(s, 4H).

Embodiment 12: the calcium ion titration of dyestuff and the constant that dissociates (Kd) method

1) the preparation of test soln:the various test solns that use the method preparation of dilution mutually to comprise 0.5 μ M calcium ion indicating dye with " zero calcium ion damping fluid " and " high-calcium ionic damping fluid ".For example, for low Kd dyestuff (

compound

2,3,6), zero calcium ion damping fluid and high-calcium ionic damping fluid are to buy calcium ion standard buffer solution test kit (Biotium, CA on market; Cat#59100).Every kind of dyestuff all prepares the test soln of 11 kinds of different calcium ionic concns.For compound 5, at first need the zero calcium ion damping fluid of preparation to comprise 10mM pH7.2MOPS, 100mM KCl and 0.5 μ M dyestuff and the high-calcium ionic damping fluid comprises 10mM pH7.2MOPS, 100mM KCl, 0.5 μ M dyestuff and 1mM CaCl ₂, then these two kinds of damping fluids dilute mutually.For example, because (: EGTA) calcium ion concn is cushioned do not contain calcium ion chelator in these two kinds of damping fluids, they only are used for preparing has than the final test solution of high-calcium ionic concentration (>=50 μ M), is used for the dyestuff with higher Kd is studied.8 kinds of final test solution that altogether prepared compound 16.

2) spectrum test: fluorescence spectrum is at room temperature tested on Hitachi F-4500, and the fluorescence spectrum of the compounds of this

invention

2,3 is used the solution of preparation respectively to repeat respectively 3 experiments under every kind of calcium ion concn.Similarly, compound 5 and compound 6 have repeated 2 experiments.

The fluorescence spectrum test result of different dyes under the different calcium ionic concn be as Fig. 1, and 3,5, shown in 7.The calculating of Kd is according to equation Log[(F – F _min)/(F _max– F)]=Log[Ca ⁺⁺] – LogK _dmiddle Log[(F – F _min)/(F _max– F)] vs.Log[Ca ⁺⁺] linearity calculated.F is at given calcium ion concn [Ca herein ⁺⁺] under fluorescence intensity, F _minat [Ca ⁺⁺the fluorescence intensity of]=0 o'clock; F _maxby the fluorescence intensity under saturated with calcium at dyestuff.The typical linear graph of dyestuff is as Fig. 2, and 4,6, shown in 8.Several typical Kd values of the compounds of this invention are listed below table 1:

Table 1

Compound	The Kd value
		Compound
2	663nM
		Compound
3	560nM
		Compound
5	672μM
		Compound
6	1.29μM

Embodiment 13:

Cultivate the HeLa cell 24 hours in 96 porocyte culture plates of black, according to " A Practical Guide to the Study of Calcium in Living Cells " (1994) Richard Nuccitelli, Ed, p165. with Castro et al. (2004) .Cell Death and Differentiation11, the description in 468-78 is carried out load by the form of dyestuff AM ester of the present invention to cell.Ion indicator AM ester prepares the storing solution of 10mM concentration with anhydrous DMSO; The storing solution of the 10mM AM ester of 1 μ L is dissolved in DMSO with 1 μ L20%Pluronic F-127() mix, and then add 1mL Krebs-Ringer-HEPES (KRH) damping fluid (136mM NaCl in mixture, 10mM HEPES, 4.7mM KCl, 1.25mM MgSO ₄, 1.25mM CaCl ₂) supplement and add 25mM glucose and 5% bovine serum albumin again.Cell is at room temperature cultivated 30 minutes with the damping fluid that contains the AM ester.Cell with the washing of KRH damping fluid for several times.Add substratum then cell to be cultivated 30 minutes so that intracellular AM ester is hydrolyzed under 37 ℃.Cell with the washing of KRH damping fluid for several times, and in the HRH damping fluid, use Olympus IX71 epifluorescence microscope and Q-Imaging Retiga2000R digital camera to develop, for the existing a kind of calcium ion indicator of Rhod-2() development, use the rhodamine filter set, or use Texas Red filter set to be developed to compound 2b and compound 12.

Before being developed in each hole, add fast the 20 μ M Calcium ionophore KRH damping fluids of 10 μ L in the damping fluid in each hole.For dyestuff consumption experiment, need in the KRH damping fluid that contains 2.8mM EGTA, to cell, be developed to cell.Do not need other processing just can be developed to cell, perhaps add 20 μ M digitonins to dissociate after the zone location of dyestuff in tenuigenin and developed, or directly developed after adding 1%Triton-X100 to be dissociated to the dyestuff compartmentation in organoid.Fluorescence in each hole is used SpectraMax Gemini fluorescence microplate reader (Applied Biosystems) to measure at the excite/emission maximum place of each dyestuff.Dyestuff compartmentation (%) can be calculated by formula ((Fd-Fb)/(Fi-Fb)) (100), Fd is the fluorescence of the cell processed with digitonin herein, Fb is the fluorescence of the cell processed with Triton X-100, and Fi is the fluorescence of untreated cell.

As shown in Figure 9, fluorography shows that the 2b dyestuff is similar (seeing the A-C of Fig. 9) in the intracellular location of HeLa to calcium ion indicator Rhod-2 with 12 dyestuffs.The cell developing that uses digitonin to process is disclosed in the cell within a cell device, has significant compartmentation effect (seeing the D-F of Fig. 9) between dyestuff and plastosome.The dyestuff that uses Triton X-100 to process is removed (TX100, be shown in the G-I of Fig. 9, and insert pictures is presented at the phase contrast fluorography of same developing regional inner cell) fully.

Show in table 2 that the relative fluorescence take off data in each hole is used for calculating dyestuff compartment rate.Demonstrate dye composition 2b and compound 12 and Rhod-2(about 30%) to compare and there is higher compartment rate (90% or higher), the compartmentation in plastosome can be found out in Fig. 9.From the experimental result of table 2 and Fig. 2, illustrate that there is obvious compartmentation in the compounds of this invention in plastosome.

Table 2

Claims

1. following logical formula I compound:

R ₅for methyl ,-CH ₂cO ₂ ^-,-L-W or

R ₆for Jia Ji Huo – CH ₂sO ₃ ^-;

L is covalently bound group;

W is carrier molecule;

2. compound as claimed in claim 1, is characterized in that,

R ₂, R ₃, R ₄be all hydrogen;

R ₅for methyl ,-CH ₂cO ₂ ^-or

Or

3. compound as claimed in claim 1, is characterized in that, R ₇for hydrogen, methyl or ethyl, R ₈for hydrogen.

4. compound as claimed in claim 1, is characterized in that, R ₇and R ₈be joined together to form six-ring.

5. compound as claimed in claim 1, is characterized in that, W is dextran.

6. following logical formula II compound:

R ₅for methyl ,-CH ₂cO ₂r ₁or

Or-CH ₂cONR ¹r ², R wherein ¹, R ²independent mutually, be hydrogen atom, C ₁-C ₁₈alkyl, C ₁-C ₁₈alkoxyl group, or by fluorine, chlorine, bromine, iodine, carboxylic acid, carboxylate salt, the C that carboxylicesters replaces ₁-C ₁₈alkyl or C ₁-C ₁₈alkoxyl group; Perhaps R ¹, R ²interconnection, directly be connected or pass through oxygen, nitrogen, sulphur, silicon is connected to form saturated or unsaturated five-ring or six-ring;

R ₆for methyl;

R ₁for methyl, ethyl or acetyl-o-methyl;

7. compound as claimed in claim 6, is characterized in that,

R ₂, R ₃, R ₄complete is hydrogen;

R ₅for methyl ,-CH ₂cO ₂r ₁or

Or

R ₁for acetyl-o-methyl.

8. compound as claimed in claim 6, is characterized in that, R ₇for hydrogen, methyl or ethyl, R ₈for hydrogen.

9. compound as claimed in claim 6, is characterized in that, R ₇with R ₈be joined together to form six-ring.

10. an application rights requires 1 described logical formula I compound to survey the method for calcium in sample ion or magnesium ion as fluorescent probe, it is characterized in that, comprises following steps:

1) sample contacts with logical formula I compound claimed in claim 1;

2) use the light source activation sample of suitable wavelength, and;

3) survey the fluorescent emission of sample.

11. method as claimed in claim 10, is characterized in that, described logical formula I compound, and in logical formula I, R ₂, R ₃, R ₄be all hydrogen; R ₅for methyl ,-CH ₂cO ₂ ^-or

Or

12. method as claimed in claim 10, is characterized in that, in step 1), if calcium ion or magnesium ion are positioned at the cell of sample, described logical formula I compound is by the microinjection transfered cell.

13. method as claimed in claim 10, is characterized in that, in step 1), if calcium ion or magnesium ion are positioned at the extracellular of sample, logical formula I compound directly contacts with sample.

14. method as claimed in claim 10, is characterized in that, in step 3), described detection is at fluorescent microscope, on microplate reader or flow cytometer device, carries out.

15. an application rights requires the method for 6 described logical formula II compounds as calcium ion or magnesium ion in fluorescent probe detection viable cell sample, comprises following steps:

2) the culturing cell sample is at least 5 minutes;

3) use the light source activation sample of suitable wavelength, and;

4) survey the fluorescent emission of sample.

16. method as claimed in claim 15, is characterized in that, described logical formula II compound, and in logical formula II, R ₂, R ₃, R ₄complete is hydrogen;

R ₅for methyl ,-CH ₂cO ₂r ₁or

Or

R ₁for acetyl-o-methyl.

17. method as claimed in claim 15, is characterized in that, described cell sample is cell culture, or the cell tissue culture.

18. method as claimed in claim 15, is characterized in that, in step 4), described detection is at fluorescent microscope, on microplate reader or flow cytometer device, carries out.